Nitric oxide and peroxynitrite production by human erythrocytes: a causative factor of toxic anemia in breast cancer patients.

In the present study we demonstrated that human erythrocytes possess a NO synthase (NOS) that can be activated by oxidative stress and Ca2+ accumulation to produce nitric oxide (NO), and that this activation could be involved in the pathogenesis of toxic anaemia in breast cancer patients. By causing oxidative stress in human erythrocytes with hydrogen peroxide (H2O2) (100 microM), or by increasing the intracellular calcium concentration using various doses (up to 100 microM) of the calcium ionophore A23187, a gradual increase in both NO and peroxynitrite (ONOO-) release that was inhibited by N-monomethyl-L-arginine (L-NMMA) (1mM) was observed. Time-dependent experiments using hemolysates showed a linear rise of NO production which was elevated by 60% in the presence of superoxide dismutase (SOD) (100 U). NOS isolated from hemolysates was constitutively expressed and was dependent on NADPH, Ca2+/calmodulin, tetrahydrobiopterin and flavins. In reconstitution experiments, when purified NOS, isolated from erythrocytes, was added to purified soluble guanylate cyclase (sGC), isolated from endothelial cells, in the presence of the appropriate cofactors and substrates, a linear increase in cGMP production at various concentrations (up to 50 microM) of H2O2 was observed. Furthermore, it was shown that erythrocytes from breast cancer patients were subjected to higher oxidative stress by ONOO- (100 microM), with a consequential increase of membrane rigidity, than erythrocytes from healthy individuals. Such mechanic changes may result in shortening of the lifespan of erythrocytes, a feature of toxic anemia in cancer patients.